Plant culture method and product

ABSTRACT

A method for reproducing large numbers of pineapple plants using a three stage plant tissue culture technique. Also the product of latent embryoid tissue (i.e., bud clusters) used in the same technique. In an induction stage, latent embryoid tissue is derived and induced to grow from an excised plant meristem. Such tissue is then grown or proliferated in an aqueous nutrient medium under conditions whereby differentiation of tissue into plantlets is inhibited. In a preferred method, the latent embryoid tissue is proliferated to a desirable amount and then transferred to a storage bank for unlimited storage. When desired, a portion of tissue may be removed from the bank and further proliferated. Also, the storage bank product. Then, in a differentiation stage, the latent embryoid tissue is caused to differentiate into plantlets.

United States Patent Corlett, Jr. et al.

[54] PLANT CULTURE METHOD AND PRODUCT [72] Inventors: Donald A. Corlett,Jr., Concord;

Panos D. Caldis, Berkeley, both of Calif.

[73] Assignee: Del Monte Corporation, San Francisco, Calif.

[22] Filed: April 1, 1970 [21] Appl. No.: 24,610

Related US. Application Data [63] Continuation-impart of Ser. No.826,445, May

21, 1969, abandoned.

52 us. Cl ..47/58 51 Int. Cl. ..A0lg 1/00 [58] Field of Search ..47/58[56] References Cited UNITED STATES PATENTS 3,514,900 6/1970 McDade..47/58 OTHER PUBLICATIONS R. Notes on the---, Kohl, Amer. Orchid Soc.Bull., February 1962, pp. 116- 120. i

S. Nutritional Requirements," Wolter et al., Amer. Journ. Botany,53(3)l966, pp. 263- 266.

[ Aug. 15, 1972 T. Mineral Oil--, Caplin, Amer. Journ. Botany, May 1959,pp. 324- 329.

U. Growth of--, Hildebrandt et a1., Amer. Journ. of

Bot., March 1963, pp. 248- 254. V. Tissue Culture, Morel, Amer. OrchidSoc. Bull., June 1964, pp. 473- 477.

Primary Examiner-Robert E. Bagwill Attorney-Flehr, l-lohbach, Test,Albritton & Herbert ABSTRACT A method for reproducing large numbers ofpineapple plants using a three stage plant tissue culture technique.Also the product of latent embryoid tissue (i.e.,'bud clusters) used inthe same technique. In an induction stage, latent embryoid tissue isderived and induced to grow from an excised plant meristem. Such tissueis then grown or proliferated in an aqueous nutrient medium underconditions whereby differentiation of tissue into plantlets isinhibited. In a preferred method, the latent embryoid tissue isproliferated to a desirable amount and then transferred to a storagebank for unlimited storage. When desired, a portion of tissue may beremoved from the bank and further proliferated. Also, the storage bankproduct. Then, in a differentiation stage, the latent embryoid tissue iscaused to differentiate into plantlets.

21 Claims, 4 Drawing Figures Patented- Aug. 15, 1972 FIG...2

INVENTORS C orle H! Donald A BY Panes D. Ceidis ATTORNEYS PLANT CULTUREMETHOD AND PRODUCT CROSS-REFERENCE TO RELATED APPLICATION Thisapplication is a continuation-in-part of copending US. application Ser.No. 826,445 in the names of 5 BACKGROUND OF THE INVENTION Many plantsare not propagated from seeds because of the genetic variability of theseed. Therefore, these plants are propagated asexually which permits themaintenance and multiplication of any genotype as clones. A commerciallyvaluable plant which is propagated asexually with shoots, slips, andcrowns is the pineapple (Ananas comosus).

Asexual propagation, while permitting generation of suitable clones, hassome severe restrictions. The most severe is that each donor plant givesonly enough plant? ing material for three to five new plants undercommercial conditions. With special techniques, more plants may beobtained, but the number is less than 40 from one plant. It may take anumber of years to produce sufficient numbers of a new hybrid or cloneto stock a plantation by vegetative propagation. In addition, since allnew plants must come from an existing previous crop, there is a greatexpenditure for retrieving the plant material and, as well, problemscreated by carryover of pests and plant diseases. Consequently, there isa need for a more satisfactory propagation technique for plants nowpropagated asexually from field material.

Tissue culture and related techniques have been used in connection withthe study and reproduction of many species of plants for many years. So,for example, in the case of strawberries, these techniques are used forthe purpose of freeing a variety from an infecting virus. So, alsoallied techniques have been used for the multiplication of meristemderived protocorms for asexual propagation of orchids and camations.These techniques, however, lead only to growth of protocormJike organsby geometrically increased numbers. It would require large storage spaceto maintain sufficient quantities of protocorms, derived from a varietyof desirable plants, to reproduce thousands of plantlets of each strainin relatively short periods of time.

Undifferentiated tissues derived from the pith of tobacco plants havebeen propagated and differentiated into plantlets under the influence ofstrong growth hormones, however, such tissues exhibit geneticinstability and often lose the ability to initiate organs. Since theplantlets are not identical to the parent, such a technique cannot beused where identical plants are needed. Still other techniques have beendeveloped for growth of large amounts of plant tissue to be consumed asfood or fodder. However, none of these techniques have been successfullyused to propagate large numbers of plants identical to the parentthrough proliferation of undifferentiated tissue.

SUMMARY OF THE INVENTION AND OBJECTS This invention relates to a plantculture technique for reproducing large numbers of plants from latentembryoid tissue (i.e., bud clusters) and to the latent embryoid tissueused in the culture technique. Three stages of the'method are:induction, proliferation, and differentiation. The latent embryoidtissue may be obtained in an induction stage preferably performed by theexcision from a donor plant of meristem tissue which is induced to growlatent embryoid tissue which, in turn, is placed in an aqueous nutrientmedium. Next, in a proliferation stage, latent embryoid tissue, whichmay be obtained in the induction stage or from other sources, isproliferated in an aqueous nutrient medium under conditions wherebydifferentiation of tissue into plantlets is inhibited. In a preferredmethod of proliferation, the latent embryoid tissue is proliferated to adesirable amount for storage and transfered to a storage bank wherein itmay be maintained for an unlimited period of time. When desired, aportion of tissue may be removed from the bank and directlydifferentiated or, if necessary, may be again proliferated. In the laststage, differentiation, the latent embryoid tissue is caused todifferentiate into plantlets which may be field planted.

It is an object of the present invention to provide an improved plantculture technique for plant reproduction.

A further object of the present invention is to propagate plants bymodified tissue culture techniques.

A further object of this invention is to make possible the developmentand reproduction of new strains of plants in a fraction of the timeheretofore required.

A further object of this invention is to produce large numbers of plantsfrom a single plant having desired characteristics.

A further object of this invention is to produce large quantities ofplants having identical characteristics which are at generally the samestage of development at the time of planting so that they may becultivated and harvested together.

It is a further object of this invention to produce a storage bank ofundifferentiated plant tissue from which tissue may be withdrawn at willand propagated to supply large quantities of plantlets.

Another object of the invention is to provide a plant culture method ofthe above character which is applicable to inducing large numbers ofpineapple plants from meristem tissue within a relative short period oftime.

A particular object of the invention is to provide tissue from pineapplemeristem from which large numbers of plants may be developed.

Further objects of this invention will be more apparent from thefollowing description taken in conjunction with the accompanyingdrawing.

BRIEF DESCRIPTION OF THE DRAWING FIGS. 1, 2, 3, and 4 are reproductionsof photographs showing van'ous stages of the product of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS According to thisinvention, a variety of plants may be propagated asexually using planttissue culture techniques. A donor plant is selected having the desiredgenetic characteristics for development into a large number of plants.Latent embryoid tissue is defined as green, granular, friable tissuewhich is genetically stable and which when grown pursuant to the planttissue culture methods of this invention is capable of proliferationwithout substantial differentiation into plantlets, and which, upon achange of culture conditions, is capable of differentiation intoplantlets. It should be understood that the granular tissue definedherein as latent embryoid tissue comprises clusters of individualpineapple buds referred to herein as bud clusters. Accordingly the twoterms are employed interchangeably in the present specification.

FIGS. 1, 2, 3, and 4 are photographs showing various development stagesof the latent embryoid tissue derived from a pineapple plant andseparated from aqueous nutrient medium. The scale, indicated in FIGS. 2and 3 by a ruler marked in centimeters and decimal portions thereof, isapproximately 2% times full size for all photographs. FIG. 1 showslatent embryoid tissue growing from remnants of originally excisedmeristem tissue derived from a mutilated bud portion 11 of a pineappleplant as the remnants appear during induction. Shoots may also developin the induction stage. Prior to the proliferation stage, meristemtissue 10 is separated from tissue 11 and from shoots having leaves 12growing therefrom. FIG. 2 shows relatively undifferentiated tissue 10 asit may appear during proliferation. Such tissue is suitably pure to bestored in a tissue bank. FIG. 3 shows partial plantlets 13 formed fromtissue 10 during the differentiation stage. FIG. 4 shows differentiatedplantlets including roots 14 and leaves 12 formed from tissue 10.

In the initial stage (i.e., induction) a portion of the plant composedof meristem tissue, such as the buds, tip of roots and leaf nodes, isexcised from the plant. In the pineapple plant, the preferred source ofmeristem tissue is the apical or lateral buds, both types being presentin the crown, and on the stem of the plant and its slips or suckers. Theleaves are removed to expose the apical and lateral buds which are thenmounted for careful removal of the bud scales, leaving one bud scale(leaf primordium) over the meristem dome. The single bud scale increasesthe survival of the mutilated meristem bud. Following the removal of thebud scales, the meristem is mutilated to modify its organization. Thisprocedure hastens the elaboration of latent embryoid tissue from the budmeristem. Simple excision of the meristem from the bud is sufficient toinduce latent embryoid tissue but induction is slow and/or sporadic.

This mutilation operation may be performed in a number of ways, as bycrushing or cutting. It is preferred to score the meristem dome withshallow cuts by small knife. By cross-cutting the dome, mutilzation isboth thorough and uniform. In a preferred mutilating technique severalshallow parallel cuts are made in a first direction across the top ofthe meristem and, then, approximately the same number of cuts are madeat right angles to the first cuts. The mutilated meristem tissue is thenplaced in an aqueous plant nutrient medium, to be described hereinafter.

Latent embryoid tissue is then induced to grow on the mutilated meristemby placing the same in the aqueous nutrient medium. Under certainconditions, growths such as shoots, rather than the desired tissue, areformed on the meristem. If so, by periodically cutting back such newgrowths (e.g., at intervals of once a week) latent embryoid tissueeventually will be induced to grow so long as the meristem remainsviable.

When the latent embryoid tissue is first formed, it is undifferentiatedbut has a strong tendency under certain conditions to differentiate intoplantlets. A critical consideration in the subsequent induction and inthe proliferation stage is the maintenance of conditions whichsubstantially inhibit differentiation of the latent embryoid tissue intoplantlets. Differentiation into plantlets is undesirable at this stagesince the plantlets absorb nutrient and seriously detract from latentembryoid tissue growth. However, as shown in FIG. 1, somedifferentiation normally occurs.

One condition to inhibit differentiation during induction andproliferation is to keep the latent embryoid tissue substantiallysubmerged in the aqueous nutrient medium. This inhibits differentiationby shielding the tissue from the atmosphere, thus preventing surfacedehydration which promotes differentiation.

Any suitable liquid nutrient medium for plant tissue culture may be usedduring induction and proliferation of which a number are known to thoseskilled in the art. Conventional media provide sources of carbohydrate(e.g., sucrose), vitamins, trace metallic elements and mineral ions(potassium, magnesium, iron, calcium, nitrate, phosphate and sulfate).In practice such aqueous media may include about 1.5 to 3 percent,preferably about 2 percent, by weight of carbohydrate along with theaforementioned vitamins an mineral ions. Although various hormones havebeen used in media available in the art, it is preferred to exclude allsuch hormones during all stages of culture of latent embryoid tissue(induction, proliferation, and tissue banking stages) since they are notnecessary to carry out the technique of this invention and could produceundesirable side effects in the latent embryoid tissue and plantlets.

An improvement of differentiation control in conventional media may beaccomplished by adjusting the iron concentration therein. It has beendiscovered that an increase in the ferrous ion concentration from theamount in conventional media, about 2.5 mg/l, two to six-fold, to about5-15 mg/l of medium, and preferably 12.5 mg/l of medium, markedlyinhibits differentiation while permitting development in mutilatedmeristem while promoting induction and development of latent embryoidtissue.

Another desirable improvement in a conventional medium is the inclusionof a polyvinylpyrrolidone such as Polycar AT produced by General Analine& Film Corporation. Polycar AT is a fine, granular, white powderinsoluble in the medium and may be added to the medium at about 50-200g/l of medium. The chief value of Polycar AT in the medium is itsability to absorb toxic phenolic compounds and browning substances fromthe nutrient medium solution. Thus, Polycar AT prevents some destructivebrowning which may occur in plant tissue cultures. It also may increasethe growth rate of latent embryoid tissue cultures by removing the toxiceffects of these browning agents.

When the pineapple is the donor plant, it is desirable to adjust thenutrient medium during induction and proliferation to a pH of between 4and 5 and, preferably, to 4.5 the pH of the natural growth of pineapplein soil. Above and below this pH, the growth rate of the latent embryoidtissue tends to decrease.

During the induction stage, it is preferred to agitate the medium inorder to circulate oxygen and the nutrients of the aqueous medium to allthe cells of the latent embryoid tissue. A reciprocal shaker or rotaryroller drum may be used.

Although light is not deemed to be necessary during induction, it ishighly desirable. The light intensity at the surface of the meristemtypically is between about 400 and about 600 foot candles regardless ofthe agitation device, although intensities as low as 200 foot candlesmay be used. Optimally 500 foot candles promotes raid growth anddevelopment of the meristem. The light source should be low in infra-redradiation, which has the undesirable effect of accentuating shootformation. Therefore, the light primarily should come from cool-whitefluorescent lamps of either the regular or high intensity type althoughfiltered sunlight or any suitable light source would be satisfactory.

With pineapple as the donor plant, growth in the nutrient duringinduction should be carried out at temperatures elevated slightly abovenormal room temperature. The range of temperatures between about 21 andC may be used. However, in order to suppress differentiation, thetemperature of new latent embryoid tissue in the flask preferably shouldbe maintained between about 23 and 245 C.

After a sufficient quantity of latent embryoid tissue has been inducedto grow on the meristem in the aqueous nutrient medium, the latentembryoid tissue is separated from the meristem and any plantlets thathave developed. Typical latent embryoid tissue at this stage is shown inFIG. 2. The separated tissue is then placed in a flask in a mediumsimilar to the one described to commence a preliminary proliferationstage. During this stage, the tissue is substantially submerged in theliquid medium to avoid direct contact with the atmosphere, andconsequent dehydration at the surface, and exposure to oxygen. Theconditions previously described are followed. By way of example, themass of the tissue in this proliferation operation may be increased tentimes or more.

Following preliminary proliferation, alternate series of steps may beperformed prior to differentiation into plantlets depending upon thedesired result. In one procedure, the total preliminary tissue ismassively proliferated as discussed hereinafter. In another procedure,the preliminary tissue is split up, a portion being placed in a storagebank, as discussed hereinafter. In still another procedure, all thepreliminary tissue is placed in a storage bank, a portion of which isremoved at a later time to supply tissue for massive proliferation.

During massive proliferation, the growth rate is substantiallyincreased. Starting portions of latent embryoid tissue are placed inseparate flasks. This is essentially a continuation of the preliminaryproliferation stage, but on a larger scale. By way of example, theincrease in the mass of tissue during massive proliferation may be ofthe order of ten times or considerably higher. However, anydifferentiated tissue, such as shoots and leaves, is removed from thetissue and only the undifferentiated, green tissue is transferred to theflasks.

The removal of differentiating tissue performed at the beginning ofproliferation, serves to inhibit further differentiation. In likemanner, any differentiating tissue is periodically removed from thetissue throughout massive proliferation to inhibit differentiation. Suchdifferentiated tissue is visually detectable when small leaf tissueappears, and may be removed manually from time to time.

During massive proliferation, the latent embryoid tissue is placed atthe bottom of a flask with sufficient liquid medium added to cover thetissue with the exception of a small portion, such as within about 2 or3 millimeters of the surface of the tissue. If large portions of thesurface of latent embryoid tissue are exposed, the tissue, especiallythe upper portion thereof, will spontaneously differentiate intoplantlets. On the other hand, if the latent embryoid tissue iscompletely or continuously submerged during massive proliferation, itgrows at a very slow rate and tends to discolor.

In practice massive proliferation of pineapple embryoid tissue has beencarried out at temperatures within the range of about 20 C to about 30C. Above 30 C, the tissue tends to brown, thus retarding proliferation.On the other hand, the growth rate is restricted beyond the lower limitof 20 C. For most vigorous growth, the optimum temperature is preferablyabout 27 C.

During massive proliferation, the lighting may be generally the same asin the induction stage although a broader range of light intensity(e.g., ZOO-1,000 foot candles) is normally employed.

As in the induction stage, some agitation to bathe the tissue withnutrient is desirable during proliferation. The agitation promotes thesupply of nutrients to the tissues, removes wastes from the tissues, andaerates the tissue. Accordingly, it is preferred to have gentleagitation on a tissue culture roller tube rotating at about one rotationper minute. Alternatively, a shaker may be used so long as there areless than about shakes per nrinute.

After massive proliferation, the tissue may be sent directly to thedifferentiation stage and caused to dif ferentiate into plantlets underconditions described hereinafter.

In an alternative procedure, instead of performing massive proliferationdirectly after preliminary proliferation, the tissue may be either allbanked (i.e., stored) followed by massive proliferation at a later timeor split with a portion being massively proliferated and a portionstored. During the storage stage, the tissue is placed in a supply bankwhich provides a means for maintaining specific latent embryoid tissuecultures of various plants indefinitely in a ready state. These latentembryoid tissue cultures are maintained in the bank under conditionswhich permit tissue which is substantially undifferentiated to grow at arelatively slow rate. A portion of the stored tissue may be removed anddifferentiated or may be proliferated in a post-storage stage and thendifferentiated. The conditions for differentiation will be describedhereinafter. The remaining portion of the tissue is then returned to thestorage bank.

To prepare a latent embryoid tissue bank for the storage stage, eachculture to be placed in the bank must be tested to verify its ability toproliferate and differentiate into plantlets. Enough green, healthylatent embryoid tissue consisting of only undifferentiated material,free from contaminating micro-organisms, is aseptically placed in thebutt of a sterile tube, e.g., a cotton stoppered, lipless, glass tube(i.e., 22 mm X cm) to fill the tube to 3 cm. Enough sterile nutrientmedium of the type previously described is aseptically pipetted into thetube to cover the tissue by about 1 cm, producing a total of about 4 cmof latent embryoid tissue and medium. After filling the tube, the tissueis allowed to incubate. These tubes having the above contents may thenbe placed at an angle on a Tissue Culture Rollerdrum (New BrunswickScientific Co., TC-4) or any suitable rack that revolves continuously atabout one revolution per minute. The tubes are incubated on the rack ata tissue temperature of about to C and preferably about 21 C undercontinuous light. The light should be produced from fluorscentcool-white" light source low in the infra-red wave length. The lightintensity preferably should be no more than about 200 foot candles onthe tubes at the top of the roller drum for slow growth although theintensity may be increased if faster growth is desired.

Plantlets, if they occur spontaneously during incubation, should beremoved at least every 2 weeks, at which time the medium should also bebrought up to about I centimeter above the latent embryoid tissue. Thelevel of the latent embryoid tissue in the tube will increase as thetissue grows.

About every month of incubation the old medium and the upper portions oflatent embryoid tissue should be removed so that only 3 centimeters ofthe tissue remains. This is necessary since the tissue approximatelydoubles every two weeks. The old medium should be removed and new mediumadded until the contents reach a 4 cm level, and the old cotton-stoppersdiscarded and replaced with new ones. This method may be used tomaintain the tissue in an undifferentiated state indefinitely.

The above quantities in the bank are meant by way of example, notlimitation. These quantities illustrate that the tissue should besubstantially submerged to inhibit differentiation.

Aseptic conditions must be scrupulously adhered to in all phases of thetissue-bank procedure. The cultures should be examinedmicro-biologically once a month when the medium is changed to detectcontamination.

One problem that might be encountered in the operation of the tissuebank is the browning and death of culture. One cause of browning resultsfrom the latent embryoid tissue becoming too packed in the base of thetube. This can be overcome by punching a hole through the latentembryoid tissue to the bottom of the tube. Another cause of browning isthe accumulation of toxic phenolic and other compounds in the nutrientsolution. As previously discussed, the addition of Polycar AT is apreventative for browning caused by these toxic compounds.

The latent embryoid tissue may be conveniently transferred from the bankto another destination by placing a portion (e.g., 10 grams) of thelatent embryoid tissue in a sterile vial, covering the tissue withnutrient medium and sealing the vial. The tissue can survive in thesevials for 4 to 5 days at room temperature in the absence of light.

With pineapple meristem, it ordinarily takes about at least 3 months toperform the induction and proliferation stages with most of the timeconsumed during induction.

The tissue treated according to this invention is bifunctional in naturein that it can grow as undifferentiated latent embryoid tissue and alsodifferentiate into plantlets. After sufficient latent embryoid tissuefor the number of required plantlets has been proliferated or after adesired amount has been withdrawn from a tissue bank and proliferated,if necessary, to the required amount, the differentiation stage iscarried out.

During the differentiation stage, it is desirable to promote the latentembryoid tissue to differentiate rapidly into plantlets. The rapidgrowth during differentiation results in a substantially synchronousmaturation and development into plantlets which may be planted over ashort period of time. When grown in the field, these plantlets yield aharvest of fruit at approximately the same stage of maturation, adesirable commercial feature.

In an optional method for assisting synchronous maturation latentembryoid tissue may be bathed with a small quantity of an aqueoussolution of a growth promoting agent, such as gibberellic acid at a lowdilution (e.g., 0.1 to pg/ml of solution). However, such treatment isnot essential.

The conditions for the differentiation stage are different from that ofeither the proliferation or the storage stages. Perhaps the mostimportant change in conditions is to expose most or a substantial partof the latent embryoid tissue to the atmosphere while a lower andrelatively minor portion of the latent embryoid tissue is in contactwith aqueous nutrient medium. In one technique this is accomplished bylowering the level of the nutrient medium in the container used forproliferation. The container may be agitated for uniform exposure oftissue to the atmosphere. Such exposure, with access to nutrients andwaste systems, causes the differentiation of latent embryoid tissue asshown in the beginning steps in FIG. 3. Maintenance of such conditionserves to promote differentiation of latent embryoid tissue intoplantlets having leaves and roots, such as in FIG. 4.

In another exposure technique, the latent embryoid tissue may bedeposited onto an aqueous agar gel having the necessary nutrients. Sincea substantial portion of the tissue is thereby open to the aunosphere,there is no need to assist uniform exposure by agitation. This techniqueassists the formation of healthy roots since the same, beinggravitropic, grow into the agar as in a natural environment. For thispurpose, the gel is preferably sufficiently rigid for support but softenough to permit root formation therein. A suitable gel concentration isabout 1.0 to 1.4 percent and preferably about 1.2 percent of agar, byweight of the medium. Sterilized soil may be employed to replace theagar with a similar effect.

During the differentiation, it has been found that the rate of forminghealthy plantlets is influenced by the nutritional content of theaqueous medium in contact with the latent embryoid tissue. If suchcontent is too high, tissue proliferation, rather than differentiation,is favored with a consequent lowering of the rate of the latter. On theother hand, if the nutrient content is too low, the rate ofdifferentiation is accelerated but at the cost of injuring a certainnumber of plantlets due to deficient nutrition. Accordingly, thenutrient concentration employed for proliferation is reduced by about 50to 90 percent, preferably 70 to 80 percent, in the differentiationmedium. Suitable differentiation media include about 0.25 to 1 percent,preferably about 0.5 percent, by weight of carbohydrate (e.g., sucrose)along with adequate vitamins and minerals.

To further promote differentiation, the conditions of light andtemperature are also changed to induce differentiation. The lightintensity may be substantially increased such as to about 1,000 footcandles or less at the tissue surface with about percent of the lightcoming from incandescent lamps. In addition, the temperature may beraised to about C.

During the differentiation stage, by the time the plantlets are about 2cm high, they have differentiated to the extend that all of thecharacteristic features of the plant are exhibited and all the plantletscan be grown in soil or other media as is currently practiced in theart. Additional growth is ordinarily necessary following differentiationbefore the plantlets are placed in the field. Under conventionalplanting conditions, shoots or suckers should weigh at least about 100gm and are 6 to 8 inches tall at the time of field planting.

As with other tissue culture techniques, it is desirable to use aseptictechniques throughout. Unless all glassware, media, pipettes, utensils,etc., are sterile, there is danger of contamination. Contamination notonly can affect the large numbers of plants ultimately differentiatedaccording to the present process it might also destroy the meristem orlatent embryoid tissue in the first instance. Glass distilled watershould be used for purity and is intended when the word water is usedherein.

EXAMPLE I A pineapple crown was cut off to remove the portion containingthe apical bud. The leaves were stripped off and the fleshy stern waswashed in a mild detergent. The top portion of the crown containing themeristem was separated and disinfected in a 10 percent bleach-90 percentcool water solution for ten minutes, and placed in a sterile container.

The tissue containing the bud was mounted to a cardboard platform andthe bud scales carefully removed until one bud scale was present overthe meristem dome. The meristem was quite small on the order of 0.25millimeters in diameter. It was then mutilated by means of shallow cutswith a small knife. Several shallow parallel cuts were made in a firstdirection across the top of the meristem and then approximately the samenumber of cuts were made at right angles to the original cuts. Thescored meristem and some of the adjacent basal tissue (about 1 cubicmillimeter) was placed in a 125 ml flask. About 6 milliliters of liquidnutrient medium was added and the flask was placed on a reciprocalshaker. The medium was prepared as follows. A double strength basal saltsolution was first of water.

Salt Amount NHJ-IO, 16.5 gm KNO, l9.0 gm CaCl,'2H,O 4.4 gm MgSO,'7H,()3.7 gm KH PO 1.7 gm 11,30, 62 mg MnSO 'H,O I69 mg ZnSO, 106 mg Kl 8.3 mlStock Solution A 5 ml Stock Solution B l ml Stock Solution C 10 ml StockSolution A is a chelating agent known as NaFeEDTA (Sequestrene, producedby Geigy Industrial Chemicals) which was prepared by adding 3.8 grams ofthe chelating agent in ml of water.

Stock Solution B was prepared by adding 25 mg of CuSOr SH O and 25 mgCoCl 6H O in 100 ml of water.

Stock Solution C was prepared by adding 25 mg of Na MoO 2H O in 100 mlof water.

The ordinary nutrient medium used in this example was prepared by adding20 grams sucrose, 100 mg meso-inositol, 500 mg casein hydrolysate and 1ml of thiamine-HCl stock solution (prepared by dissolving 50 mgthiamine-HC] in 100 ml water) to 500 ml of the double strength basalsalt solution. The pH was adjusted to 4.5 using 0.1 N hydrochloric acid.The final volume of the medium was adjusted to l L with water.

The scored meristem, together with some adjacent basal tissue was placedin the foregoing nutrient media and agitated on a reciprocal shaker at atemperature between 25 and 27 C. The flask was subject to constantillumination from fluorescent light (500 foot candles, noincandescence). Low infra-red producing fluorescent lights wereemployed. The medium was changed every 7 days.

The scored meristem was permitted to grow in the nutrient medium forseveral weeks. After 4-6 weeks, the masses of budding tissue weredivided up into four separate parts and again placed in 6 ml of nutrientmedia and subjected to further agitation under constant illumination. Assmall plantlets were developed throughout this step, they were separatedand discarded. The uniform, undifferentiated latent embryoid tissue,green in color, was permitted to develop in the medium by the removal ofthe small plantlets.

After the development of an appreciable quantity, such as 1 or 2 gramsof the latent embryoid tissue, it was placed in a ml flask containing 10ml of nutrient medium.

During proliferation, the latent embryoid tissue was constantly agitatedand illuminated in the nutrient medium until the bottom of the 125 mlflask was covered by a layer about '2 or 3 cm thick. This tissue wasthen transferred to larger flasks and grown further by the sametechnique until the desired quantity of latent embryoid tissue wasobtained. The liquid level of the medium was kept within 2 or 3 mm ofthe surface of the tissue to inhibit spontaneous differentiation intoplantlets without causing browning of the tissue. The occasionalplantlets that were formed were weeded out whenever the medium waschanged. The medium was changed each week.

When it was desired to differentiate the latent embryoid tissue intoplantlets, gibberellic acid in the amount of 10 ug/ml was added to themedium. The tissue was bathed under substantially submerged conditionsat a temperature of about 28 C to about 30 C under constant illuminationby daylight-simulating fluorescent lights (1,000 foot candles, 10percent incandescence). At the end of 3 days the medium, containing thegibberellic acid, was drained and the tissue was spread in a layer about1 cm thick in a 500 ml Erlenmeyer flask. Four ml of medium, absentgibberellic acid was added and the flask was again agitated under theconditions of temperature and light as described with respect to thegibberellic acid medium. The consumption and evaporation of the nutrientmedium required the addition of 4 ml at least every 7 days. In 8 weeks,plantlets measuring 2 cm in height were formed which were planted insoil in the laboratory or other controlled environment. After theseplantlets had grown to a height of about 6 to 8 inches, they were readyfor planting in the field.

Because all the plantlets were growing from a mass of latent embryoidtissue produced at the same time and because of the effect of thegrowth-promoting agent, gibberellic acid, the stage of development ofthe plants was relatively similar so that planting conveniently tookplace over a short period of time with the result that pineapple plantswill bear fruit simultaneously.

The differentiation in the plants was also carried out on the surface ofsolid agar. Any suitable nutrient medium was used, but satisfactoryresults have been found using I-Iills Orchid Culture Medium of unknowncomposition, known to those in the orchid-growing art. No agitation wasemployed. The flasks were incubated at 283 at 5001,000 foot candles oflight.

EXAMPLE 2 Using the general procedure of Example 1, large scale plantletproduction was carried out. The procedure consists of three steps: thefirst two for rapidly proliferating latent embryoid tissue and the thirdfor large scale proliferation, differentiation and plantlet maturation.

In step 1, one ounce of latent embryoid tissue from pineapple meristemwas proliferated into 2 ounces in 7 days.

In step 2, the 2 ounce quantity of latent embryoid tissue obtained bystep 1 was divided into two flasks, 1 ounce per flask, whereupon it wasproliferated into a total quantity of ounces in 10 days.

In step 3, 5 ounces of latent embryoid tissue, obtained by steps 1 and 2in 17% days, was then divided evenly into 10 flasks (500 ml), one-halfounce per flask. The latent embryoid tissue was present in each flask.The rate of weight increase was approximately constant.

On the 40th day conditions were changed to favor differentiation (i.e.,low liquid level, temperature increase, and increase in light'intensity,especially in the infra-red wavelengths).

Differentiation occurred between 40 and 45 days. The rate of weight gainthe flasks changed from the 42d day on, indicating the growth ofplantlets instead of latent embryoid tissue. Difierentiation occurred atthe end of 62 days, at which time the tissue weight averaged 33% ouncesper flask. The 10 flasks yielded a total of 10,000 plantlets.

The quantitive aspects of producing 10,000 plants from an initial lounce quantity of latent embryoid tissue are outlined in Table I.

The growth or proliferation rate of latent embryoid tissue is slowerthan the growth rate of plantlets. One reason is that although theinitial growth rate of latent embryoid tissue is high, it slows down dueto lack of physical space. After differentiation occurs, there is afinite number of plants which are able to grow upward. This is thereason the initial latent embryoid tissue proliferation is carried outstepwise in increasing numbers of flasks with frequent changes ofmedium.

EXAMPLE III The procedure of Example I was essentially followed duringinduction and proliferation with the following modifications duringdifferentiation. Treatment with gibberellic acid was omitted as notbeing considered necessary for synchronous differentiation. The tissuewas transferred from the ml proliferation flask and placed in a uniformmanner onto the surface of a soft aqueous agar gel containing a nutrientmedium of the same type as employed for proliferation, but diluted toone-quarter strength by the addition of water. The agar was present inthe amount 1.2 percent by weight of the nutrient medium solution. Thetissue was substantially entirely exposed to the surrounding air with aminor lower portion in contact with, and deriving nourishment from themedium in the agar. The flasks were incubated at about 2830 C atSOD-1,000 foot candles of light with no agitation. The medium wasreplaced about as frequently as in Example 1. By employing the abovetechnique, the yield of healthy usable plantlets was higher than thatobtained from either of the differentiation methods of Example I.

With respect to the differentiating technique used in Example I, it wasnoted that while gibberellic acid accelerated differentiation, thepineapple plantlets tended to be spindly. Such plants may have a seriousmortality rate when transferred to soil. Also when using agar gel as inExample I, it was noted that the plants during growth tended to rise upabove the gel surface and that this tended to slow down the growth rate,probably because this reduced the ability of the plant to utilize thenutrients of the gel. Also it was noted in Example I that the HillsOrchid Medium tended to produce relatively dry exposed surfaces whichare believed to cause production of a higher percentage of unhealthy andunusable plants. The method of Example III overcame the foregoingdifficulties and produced a high percentage of healthy plants whichcould be readily transferred to soil for further growth.

The above plant tissue culture technique produces many advantages. Thetime taken to grow various strains of plants and to grow crops fromcommercial plants is substantially shortened. Over 10,000 plantlets maybe reproduced in 2 months starting with 1 ounce of tissue from the bank.Banks of tissue culture of desirable strains may be maintained and drawnupon to supply a source of tissue for proliferation and differentiationinto plantlets at a desired time for planting. Furthermore, thecharacteristics of the plant are reproduced directly from an existingplant of desired characteristics free of contaminants.

We claim:

I. A method for producing a pineapple plant culture comprising removingmeristem tissue from a donor plant, incising the meristem tissue topromote growth of bud clusters therefrom, said growth occurring whilethe meristem is in aqueous nutrient medium, and subjecting the budclusters to predetermined environmental conditions in an aqueousnutrient medium to proliferate the bud clusters without substantialdifferentiation into plantlets.

2. A method as in claim 1 in which the promoted bud clusters areseparated from any remaining donor tissue prior to proliferation.

3. A method as in claim 1 in which the bud clusters are subjected tomodified environmental conditions to cause the same to differentiateinto plantlets.

4. A method as in claim 1 wherein the meristem tissue removed from thedonor plant is a bud meristem.

5. A method as in claim 1 wherein the aqueous nutrient medium contains acarbohydrate, vitamins,

' trace metallic elements, and ions of potassium, magnesium, calcium,nitrate, phosphate, sulfate, and chelated ferrous ion.

6. A method as in claim 1 together with the steps of storing theproliferated bud clusters in a container bank with sufficient aqueousnutrient medium to substantially cover the bud clusters, maintaining thecontainer bank at conditions whereby differentiation into plantlets isinhibited, agitating the container bank, removing the medium and upperportion of the bud clusters periodically and adding sufficient newmedium to again cover the remaining bud clusters.

7. A method as in claim 1 wherein the aqueous nutrient medium containsvitamins, a carbohydrate, trace metallic elements and ions of potassium,magnesium, calcium, nitrate, phosphate, sulfate, and at least about 5mg/l of chelated ferrous ion and polyvinylpyrrolidone in the quantity ofabout 50-100 mg/l of medi- 8. A method for reproducing large numbers ofpineapple plants from pineapple bud clusters comprising subjecting saidbud clusters to predetermined environmental conditions whilesubstantially submerged in an aqueous nutrient medium to proliferate thebud clusters into a mass without substantial differentiation intoplantlets, and thereafter exposing a substantial upper surface portionof the proliferated bud cluster mass to the atmosphere andsimultaneously contacting a lower surface of the bud cluster mass withaqueous nutrient medium to cause an upper segment of the roliferated budclusters to ifferentiate into tl ts. p 9. A method as in claim gwhereinsaid m ii ii m employed for differentiation includes substantially thesame type of nutrient as the medium employed for proliferation.

10. A method as in claim 9 in which the nutrient concentration in theproliferation medium is reduced by about 50 to 90 percent in thedifferentiation medium.

11. A method as in claim 9 wherein the nutrient concentration is reducedby about to percent in the differentiation medium.

12. A method as in claim 9 in which the nutrient medium includescarbohydrates and trace metallic elements, and ions of iron, potassium,magnesium, calcium, nitrate, phosphate and sulfate.

13. A method as in claim 8 in which the proliferation medium nutrientsinclude about 1.5 to 3 percent by weight of carbohydrate along withadequate vitamins and minerals.

14. A method as in claim 8 in which the differentiation medium nutrientsinclude 0.25 to 1 percent by weight of carbohydrate along with adequatevitamins and minerals.

15. A method as in claim 8 wherein differentiation into plantlets isperformed on a soft inert gel containing aqueous nutrient medium.

16. A method as in claim 15 wherein the gel is formed of agar dispersedin the aqueous nutrient medium, said agar being present in a quantity ofabout 1 to 1.4 percent of the medium.

17. A culture comprising granular pineapple bud clusters free-livingapart from the donor pineapple plant, at least a portion of the budclusters being in contact with aqueous nutrient, said bud clusters beingcharacterized by the capacity to proliferate to form more granuleswithout substantial differentiation into plantlets under firstpredetermined environmental conditions and the capacity to differentiateinto a plurality of pineapple plantlets by modifying the environmentalconditions.

18. A culture as in claim 17 in which the first environmental conditionscomprise submergence in liquid nutrient medium and the secondenvironmental conditions comprise exposure to an oxygen-containingenvironment.

19. A culture as in claim 17 in which substantially all of the budclusters are submerged in said medium.

20. A culture as in claim 19 in which an upper surface portion of thebud clusters is exposed to the atmosphere and a lower surface portion ofthe same is in contact with aqueous nutrient medium.

21. A culture as in claim 20 in which a soft inert gel is dispersed inthe aqueous nutrient medium and a lower surface portion of the budclusters rests on the agar in contact with the medium while an uppersurface portion of the bud clusters is exposed to the atmosphere.

1. A method for producing a pineapple plant culture comprising removingmeristem tissue from a donor plant, incising the meristem tissue topromote growth of bud clusters therefrom, said growth occurring whilethe meristem is in aqueous nutrient medium, and subjecting the budclusters to predetermined environmental conditions in an aqueousnutrient medium to proliferate the bud clusters without substantialdifferentiation into plantlets.
 2. A method as in claim 1 in which thepromoted bud clusters are separated from any remaining donor tissueprior to proliferation.
 3. A method as in claim 1 in which the budclusters are subjected to modified environmental conditions to cause thesame to differentiate into plantlets.
 4. A method as in claIm 1 whereinthe meristem tissue removed from the donor plant is a bud meristem.
 5. Amethod as in claim 1 wherein the aqueous nutrient medium contains acarbohydrate, vitamins, trace metallic elements, and ions of potassium,magnesium, calcium, nitrate, phosphate, sulfate, and chelated ferrousion.
 6. A method as in claim 1 together with the steps of storing theproliferated bud clusters in a container bank with sufficient aqueousnutrient medium to substantially cover the bud clusters, maintaining thecontainer bank at conditions whereby differentiation into plantlets isinhibited, agitating the container bank, removing the medium and upperportion of the bud clusters periodically and adding sufficient newmedium to again cover the remaining bud clusters.
 7. A method as inclaim 1 wherein the aqueous nutrient medium contains vitamins, acarbohydrate, trace metallic elements and ions of potassium, magnesium,calcium, nitrate, phosphate, sulfate, and at least about 5 mg/l ofchelated ferrous ion and polyvinylpyrrolidone in the quantity of about50-100 mg/l of medium.
 8. A method for reproducing large numbers ofpineapple plants from pineapple bud clusters comprising subjecting saidbud clusters to predetermined environmental conditions whilesubstantially submerged in an aqueous nutrient medium to proliferate thebud clusters into a mass without substantial differentiation intoplantlets, and thereafter exposing a substantial upper surface portionof the proliferated bud cluster mass to the atmosphere andsimultaneously contacting a lower surface of the bud cluster mass withaqueous nutrient medium to cause an upper segment of the proliferatedbud clusters to differentiate into plantlets.
 9. A method as in claim 8wherein said medium employed for differentiation includes substantiallythe same type of nutrient as the medium employed for proliferation. 10.A method as in claim 9 in which the nutrient concentration in theproliferation medium is reduced by about 50 to 90 percent in thedifferentiation medium.
 11. A method as in claim 9 wherein the nutrientconcentration is reduced by about 70 to 80 percent in thedifferentiation medium.
 12. A method as in claim 9 in which the nutrientmedium includes carbohydrates and trace metallic elements, and ions ofiron, potassium, magnesium, calcium, nitrate, phosphate and sulfate. 13.A method as in claim 8 in which the proliferation medium nutrientsinclude about 1.5 to 3 percent by weight of carbohydrate along withadequate vitamins and minerals.
 14. A method as in claim 8 in which thedifferentiation medium nutrients include 0.25 to 1 percent by weight ofcarbohydrate along with adequate vitamins and minerals.
 15. A method asin claim 8 wherein differentiation into plantlets is performed on a softinert gel containing aqueous nutrient medium.
 16. A method as in claim15 wherein the gel is formed of agar dispersed in the aqueous nutrientmedium, said agar being present in a quantity of about 1 to 1.4 percentof the medium.
 17. A culture comprising granular pineapple bud clustersfree-living apart from the donor pineapple plant, at least a portion ofthe bud clusters being in contact with aqueous nutrient, said budclusters being characterized by the capacity to proliferate to form moregranules without substantial differentiation into plantlets under firstpredetermined environmental conditions and the capacity to differentiateinto a plurality of pineapple plantlets by modifying the environmentalconditions.
 18. A culture as in claim 17 in which the firstenvironmental conditions comprise submergence in liquid nutrient mediumand the second environmental conditions comprise exposure to anoxygen-containing environment.
 19. A culture as in claim 17 in whichsubstantially all of the bud clusters are submerged in said medium. 20.A culture as in claim 19 in which an upper surface portion of the budclusters is expoSed to the atmosphere and a lower surface portion of thesame is in contact with aqueous nutrient medium.
 21. A culture as inclaim 20 in which a soft inert gel is dispersed in the aqueous nutrientmedium and a lower surface portion of the bud clusters rests on the agarin contact with the medium while an upper surface portion of the budclusters is exposed to the atmosphere.